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The cytokine interleukin-21 (IL-21) is closely related to IL-2 and IL-15, and their receptors all share the common cytokine receptor γ chain, [γ.sub.c], which is mutated in humans with X-linked severe combined immunodeficiency disease (XSCID). We demonstrate that, although mice deficient in the receptor for IL-21 (IL-21R) have normal lymphoid development, after immunization, these animals have higher production of the immunoglobulin IgE, but lower IgG1, than wild-type animals. Mice lacking both IL-4 and IL-21R exhibited a significantly more pronounced phenotype, with dysgammaglobulinemia, characterized primarily by a severely impaired IgG response. Thus, IL-21 has a significant influence on the regulation of B cell function in vivo and cooperates with IL-4. This suggests that these [γ.sub.c]-dependent cytokines may be those whose inactivation is primarily responsible for the B cell defect in humans with XSCID., The receptor for the lymphoid-specific cytokine IL-21 is expressed on T, B, and NK cells (1, 2). IL-21 was initially reported to have a costimulatory T cell proliferative effect, to [...]

I have performed genetic studies on murine acquired immunodeficiency syndrome (MAIDS) susceptibility that suggest an alternative explanation for the results reported by O. Kanagawa et al. (1). My results raise [...]

The development of an immunodeficiency syndrome of mice caused by a replication-defective murine leukemia virus (MuLV) is paradoxically associated with a rapid activation and proliferation of [CD4.sup.+] T cells that [...]

The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed IrfS and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells. [ABSTRACT FROM AUTHOR]

B cells possess various immuno regulatory functions. However, research about their participation in tolerance induction toward the fetus is just emerging. Accumulating evidence supports the idea that B cells can play seemingly conflicting roles during pregnancy, either protecting or harming the fetus. Previous findings indicated the presence of two different peritoneal B cell subsets, defined by the expression of the plasma cell alloantigen 1 (PC1) and with distinct immune modulatory functions. Here, we aimed to study the participation of these two B cell subsets, on pregnancy outcome in a murine model of disturbed fetal tolerance. The frequencies and cell numbers of peritoneal and splenic CD19+IL-10+ and CD19+CD5+IL-10+PC1+ cells were assessed in virgin as well as normal pregnant (NP) and abortion-prone (AP) females during the course of gestation. Peritoneal PC1low or PC1high B1a B cells were sorted, analyzed for their ability to secrete IL-10 and adoptively transferred into NP or AP females. On gestation day (gd) 12, the abortion rate as well as the frequencies and cell numbers of regulatory T cells, TH1 and TH17 cells were determined in spleens and decidua. In addition, mRNA expression of IL-10, TGF-β, IFN-γ, and TNF-α was analyzed in decidual tissue. Peritoneal CD19+IL-10+ and CD19+CD5+IL-10+PC1+ frequencies fluctuated during the progression of normal pregnancies while no significant changes were observed in spleen. AP females showed significantly reduced frequencies of both B cell populations and exhibited an altered peritoneal PC1high/PC1low ratio at gd10. Adoptive transfers of PC1low B1a B cells into NP females increased the abortion rate in association with a reduced splenic regulatory T/TH17 ratio. By contrast, the transfer of PC1high B1a B cells into AP females significantly diminished the fetal rejection rate and significantly reduced the numbers of splenic TH17 cells. Our results suggest that the peritoneum harbors two distinct B1a B cell subsets that can be distinguished by their PC1 expression. Whereas PC1high B1a B cells seem to support fetal survival, PC1low cells B1a B cells may compromise fetal well-being. [ABSTRACT FROM AUTHOR]

Allelic Exclusion in Transgenic Mice That Express the Membrane Form of Immunoglobulin μ REGULATION OF IMMUNOGLOBULINgene expression involves genomic rearrangements at a minimum of two loci that bring regulatory and [...]

Most natural IgM antibodies are encoded by germline Ig sequences and are produced in large quantities by both mice and humans in the absence of intentional immunization. Natural IgM are reactive with many conserved epitopes, including those shared by microorganisms and autoantigens. As a result, these antibodies play important roles in clearing intruding pathogens, as well as apoptotic/necrotic cells and otherwise damaged tissues. While natural IgM binds to target structures with low affinity due to a lack of significant selection by somatic hypermutation, its pentameric structure with 10 antigen-binding sites enables these antibodies to bind multivalent target antigens with high avidity. Opsonization of antigen complexed with IgM is mediated by cell surface Fc receptors. While the existence of Fc alpha/mu receptor has been known for some time, only recently has the Fc receptor specific for IgM (FCMR) been identified. In this review, we focus on our current understandings of how natural IgM and FCMR regulate the immune system and maintain homeostasis under physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

Background: Protein kinase A (PKA) is a holoenzyme that consists of a dimer of regulatory subunits and two inactive catalytic subunits that bind to the regulatory subunit dimer. Four regulatory subunits (RIa, RIβ, RIIa, RIIβ) and four catalytic subunits (Ca, Cβ, C, Prkx) have been described in the human and mouse genomes. Previous studies showed that complete inactivation of the Prkar1a subunit (coding for RIa) in the germline leads to embryonic lethality, while Prkar1a-deficient mice are viable and develop schwannomas, thyroid, and bone neoplasms, and rarely lymphomas and sarcomas. Mice with inactivation of the Prkar2a and Prkar2b genes (coding for RIIa and RIIβ, respectively) are also viable but have not been studied for their susceptibility to any tumors. Methods: Cohorts of Prkar1a+/-, Prkar2a+/-, Prkar2a-/-, Prkar2b+/- and wild type (WT) mice have been observed between 5 and 25 months of age for the development of hematologic malignancies. Tissues were studied by immunohistochemistry; tumor-specific markers were also used as indicated. Cell sorting and protein studies were also performed. Results: Both Prkar2a-/- and Prkar2a+/- mice frequently developed hematopoietic neoplasms dominated by histiocytic sarcomas (HS) with rare diffuse large B cell lymphomas (DLBCL). Southern blot analysis confirmed that the tumors diagnosed histologically as DLBCL were clonal B cell neoplasms. Mice with other genotypes did not develop a significant number of similar neoplasms. Conclusions: Prkar2a deficiency predisposes to hematopoietic malignancies in vivo. RIIa's likely association with HS and DLBCL was hitherto unrecognized and may lead to better understanding of these rare neoplasms. [ABSTRACT FROM AUTHOR]

Avian v-myc Replaces Chromosomal Translocation in Murine Plasmacytomagenesis PLASMACYTOMAS CAN BE REGULARLYinduced in inbred BALB/cAn mice by the intraperitoneal (i.p.) injection of pristane (2,6,10,14-tetramethylpentadecane) (1). The incidence of plasmacytomas varies [...]

Background: The crisis of Misidentified and contaminated cell lines have plagued the biological research community for decades. Some repositories and journals have heeded calls for mandatory authentication of human cell lines, yet misidentification of mouse cell lines has received little publicity despite their importance in sponsored research. Short tandem repeat (STR) profiling is the standard authentication method, but it may fail to distinguish cell lines derived from the same inbred strain of mice. Additionally, STR profiling does not reveal karyotypic changes that occur in some high-passage lines and may have functional consequences. Single nucleotide polymorphism (SNP) profiling has been suggested as a more accurate and versatile alternative to STR profiling; however, a high-throughput method for SNP-based authentication of mouse cell lines has not been described. Results: We have developed computational methods (Cell Line Authentication by SNP Profiling, CLASP) for cell line authentication and copy number analysis based on a cost-efficient SNP array, and we provide a reference database of commonly used mouse strains and cell lines. We show that CLASP readily discriminates among cell lines of diverse taxonomic origins, including multiple cell lines derived from a single inbred strain, intercross or wild caught mouse. CLASP is also capable of detecting contaminants present at concentrations as low as 5%. Of the 99 cell lines we tested, 15 exhibited substantial divergence from the reported genetic background. In all cases, we were able to distinguish whether the authentication failure was due to misidentification (one cell line, Ba/F3), the presence of multiple strain backgrounds (five cell lines), contamination by other cells and/or the presence of aneuploid chromosomes (nine cell lines). Conclusions: Misidentification and contamination of mouse cell lines is potentially as widespread as it is in human cell culture. This may have substantial implications for studies that are dependent on the expected background of their cell cultures. Laboratories can mitigate these risks by regular authentication of their cell cultures. Our results demonstrate that SNP array profiling is an effective method to combat cell line misidentification. [ABSTRACT FROM AUTHOR]

Nfatc2 and Tob1 are intrinsic negative regulators of T cell activation. Nfatc2-deficient and Tob1-deficient T cells show reduced thresholds of activation; however, whether these factors have independent or overlapping roles in negative regulation of T cell responses has not been previously examined. Here, we show that Nfatc2 knockout (KO) but not Tob1 KO mice have age-associated accumulation of persistently activated T cells in vivo and expansion of the CD44+ memory cell compartment and age-associated lymphocytic infiltrates in visceral organs, without significant changes in numbers of CD4+CD25+Foxp3+ regulatory T cells (Treg). In vitro, CD4+CD25− “conventional” T cells (Tconvs) from both KO strains showed greater proliferation than wild type (WT) Tconvs. However, while Tregs from Nfatc2 KO mice retained normal suppressive function, Tregs from Tob1 KOs had enhanced suppressive activity. Nfatc2 KO Tconvs expanded somewhat more rapidly than WT Tconvs under conditions of homeostatic proliferation, but their accelerated growth capacity was negated, at least acutely, in a lymphoreplete environment. Finally, Nfatc2 KO mice developed a previously uncharacterized increase in B-cell malignancies, which was not accelerated by the absence of Tob1. The data thus support the prevailing hypothesis that Nfatc2 and Tob1 are non-redundant regulators of lymphocyte homeostasis. [ABSTRACT FROM AUTHOR]

This study aimed to determine whether the multi-kinase inhibitor dasatinib would provide an effective therapy for myeloproliferative diseases (MPDs) involving c-Cbl mutations. These mutations, which occur in the RING finger and linker domains, abolish the ability of c-Cbl to function as an E3 ubiquitin ligase and downregulate activated protein tyrosine kinases. Here we analyzed the effects of dasatinib in a c-Cbl RING finger mutant mouse that develops an MPD with a phenotype similar to the human MPDs. The mice are characterized by enhanced tyrosine kinase signaling resulting in an expansion of hematopoietic stem cells, multipotent progenitors and cells within the myeloid lineage. Since c-Cbl is a negative regulator of c-Kit and Src signaling we reasoned that dasatinib, which targets these kinases, would be an effective therapy. Furthermore, two recent studies showed dasatinib to be effective in inhibiting the in vitro growth of cells from leukemia patients with c-Cbl RING finger and linker domain mutations. Surprisingly we found that dasatinib did not provide an effective therapy for c-Cbl RING finger mutant mice since it did not suppress any of the hematopoietic lineages that promote MPD development. Thus we conclude that dasatinib may not be an appropriate therapy for leukemia patients with c-Cbl mutations. We did however find that dasatinib caused a marked reduction of pre-B cells and immature B cells which correlated with a loss of Src activity. This study is therefore the first to provide a detailed characterization of in vivo effects of dasatinib in a hematopoietic disorder that is driven by protein tyrosine kinases other than BCR-ABL. [ABSTRACT FROM AUTHOR]

La antigen (Sjögren's syndrome antigen B) is a phosphoprotein associated with nascent precursor tRNAs and other RNAs, and it is targeted by autoantibodies in patients with Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Increased levels of La are associated with leukemias and other cancers, and various viruses usurp La to promote their replication. Yeast cells (Saccharomyces cerevisiae and Schizosaccharomyces pombe) genetically depleted of La grow and proliferate, whereas deletion from mice causes early embryonic lethality, raising the question of whether La is required by mammalian cells generally or only to surpass a developmental stage. We developed a conditional La allele and used it in mice that express Cre recombinase in either B cell progenitors or the forebrain. B cell Mb1Cre La-deleted mice produce no B cells. Consistent with αCamKII Cre, which induces deletion in hippocampal CA1 cells in the third postnatal week and later throughout the neocortex, brains develop normally in La-deleted mice until ∼5 weeks and then lose a large amount of forebrain cells and mass, with evidence of altered pre-tRNA processing. The data indicate that La is required not only in proliferating cells but also in nondividing postmitotic cells. Thus, La is essential in different cell types and required for normal development of various tissue types. [ABSTRACT FROM AUTHOR]

The p53 tumor suppressor exerts a central role in protecting cells from oncogenic transformation. Accordingly, the p53 gene is mutated in a large number of human cancers. In mice, germ-line inactivation of p53 confers strong predisposition to development of different types of malignancies, but the early onset of thymic lymphomas in the majority of the animals prevents detailed studies of tumorigenesis in other tissues. Here, we use the Cre/Lox approach to inactivate p53 in mature B cells in mice (referred to as "CP" B cells) and find that such p53 inactivation results in the routine development of IgM-positive CP peripheral B-cell lymphomas. The CP lymphomas generally appear to arise, even in mice subjected to immunization protocols to activate germinal center reaction, from naive B cells that had not undergone immunoglobulin (Ig) heavy chain gene class switching or somatic hypermutation. In contrast to thymic lymphomas that arise in p53-deficient mice, which generally lack clonal translocations, nearly all analyzed CP B-cell tumors carried clonal translocations. However, in contrast to spontaneous translocations in other mouse B-cell tumor models, CP B-cell tumor translocations were not recurrent and did not involve Ig loci. Therefore, CP tumors might provide models for human lymphomas lacking Ig translocations, such as splenic marginal zone B-cell lymphoma or Waldenstrom macroglobulinemia. Our studies indicate that deletion of p53 is sufficient to trigger transformation of mature B cells and support the notion that p53 deficiency may allow accumulation of oncogenic translocations in B cells. [ABSTRACT FROM AUTHOR]

Innate-like B-1a cells contribute significantly to circulating natural antibodies and mucosal immunity as well as to immunoregulation. Here we show that these classic functions of B-1a cells segregate between two unique subsets defined by expression of plasma cell alloantigen 1 (PC1), also known as ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1). These subsets, designated B-1a. PC1lo and B-1a.PC1hi, differ significantly in IgH chain utilization. Adoptively transferred PC1lo cells secreted significantly more circulating natural IgM and intestinal IgA than PC1hi cells. In contrast, PC1hi cells produced more IL-10 than PC1lo cells when stimulated with LPS and phorbol 12-myristate 13-acetate (PMA). PC1hi cells were also more efficient than PC1lo cells in regulating Th1 cell differentiation, even though both B-1a subsets were comparably active in stimulating T-cell proliferation. Furthermore, PC1lo cells generated antigen-specific IgM responses to pneumococcal polysaccharide antigens, whereas PC1hi cells do not. We found that PC1hi cells develop from an early wave of B-1a progenitors in fetal life, whereas ~ cells are generated from a later wave afterbirth. We conclude that identification of B-1a.PC1lo and B-1a.PC1hi cells extends the concept of a layered immune system with important implications for developing effective vaccines and promoting the generation of immunoregulatory B cells. [ABSTRACT FROM AUTHOR]

Many tumors are characterized by recurrent translocations between a tissue-specific gene and a proto-oncogene. The juxtaposition of the Ig heavy chain gene and Myc in Burkitt's lymphoma and in murine plasmacytoma is a classic example. Regulatory elements within the heavy chain constant region locus are required for Myc translocation and/or deregulation. However, many genes are regulated by cis-acting elements at distances up to 1,000 kb outside the locus. Such putative distal elements have not been examined for the heavy chain locus, particularly in the context of Myc translocations. We demonstrate that a transgene containing the Ig heavy chain constant region locus, inserted into five different chromosomal locations, can undergo translocations involving Myc. Furthermore, these translocations are able to generate plasmacytomas in each transgenic line. We conclude that the heavy chain constant region locus itself includes all of the elements necessary for both the translocation and the deregulation of the proto-oncogene. [ABSTRACT FROM AUTHOR]

Human Burkitt lymphomas are divided into two main clinical variants: the endemic form, affecting African children infected with malaria and the Epstein-Barr virus, and the sporadic form, distributed across the rest of the world. However, whereas sporadic translocations decapitate Myc from 5' proximal regulatory elements, most endemic events occur hundreds of kilobases away from Myc. The origin of these rearrangements and how they deregulate oncogenes at such distances remain unclear. We here recapitulate endemic Burkitt lymphoma-like translocations in plasmacytomas from uracil /V-glycosylase and activation-induced cytidine deaminase-deficient mice. Mapping of translocation breakpoints using an acetylated histone H3 lysine 9 chromatin immunoprecipitation sequencing approach reveals Igh fusions up to ∼350 kb upstream of Myc or the related oncogene Mycn. A comprehensive analysis of epigenetic marks, Polll recruitment, and transcription in tumor cells demonstrates that the 3' Igh enhancer (Ect) vastly remodels ∼450 kb of chromatin into translocated sequences, leading to significant polymerase occupancy and constitutive oncogene expression. We show that this long-range epigenetic reprogramming is directly proportional to the physical interaction of Ect with translocated sites. Our studies thus uncover the extent of epigenetic remodeling by Ig 3' enhancers and provide a rationale for the long-range deregulation of translocated oncogenes in endemic Burkitt lymphomas. The data also shed light on the origin of endemic:like chromosomal rearrangements. [ABSTRACT FROM AUTHOR]

Transcriptional activation of MYC is a hallmark of many B cell lineage neoplasms. MYC provides a constitutive proliferative signal but can also initiate ARF-dependent activation of p53 and apoptosis. The E3 ubiquitin ligase, ARF-BP1, encoded by HUWE1, modulates the activity of both the MYC and the ARF-p53 signaling pathways, prompting us to determine if it is involved in the pathogenesis of MYC-driven B cell lymphomas. ARF-BP1 was expressed at high levels in cell lines from lymphomas with either wild type or mutated p53 but not in ARF-deficient cells. Downregulation of ARF-BP1 resulted in elevated steady state levels of p53, growth arrest and apoptosis. Co-immunoprecipitation studies identified a multiprotein complex comprised of ARF-BP1, ARF, p53, MYC and the multifunctional DNA-binding factor, CTCF, which is involved in the transcriptional regulation of MYC, p53 and ARF. ARF-BP1 bound and ubiquitylated CTCF leading to its proteasomal degradation. ARF-BP1 and CTCF thus appear to be key cofactors linking the MYC proliferative and p53-ARF apoptotic pathways. In addition, ARF-BP1 could be a therapeutic target for MYC-driven B lineage neoplasms, even if p53 is inactive, with inhibition reducing the transcriptional activity of MYC for its target genes and stabilizing the apoptosis-promoting activities of p53. [ABSTRACT FROM AUTHOR]

Background: BORIS/CTCFL is a paralogue of CTCF, the major epigenetic regulator of vertebrate genomes. BORIS is normally expressed only in germ cells but is aberrantly activated in numerous cancers. While recent studies demonstrated that BORIS is a transcriptional activator of testis-specific genes, little is generally known about its biological and molecular functions. Methodology/Principal Findings: Here we show that BORIS is expressed as 23 isoforms in germline and cancer cells. The isoforms are comprised of alternative N- and C-termini combined with varying numbers of zinc fingers (ZF) in the DNA binding domain. The patterns of BORIS isoform expression are distinct in germ and cancer cells. Isoform expression is activated by downregulation of CTCF, upregulated by reduction in CpG methylation caused by inactivation of DNMT1 or DNMT3b, and repressed by activation of p53. Studies of ectopically expressed isoforms showed that all are translated and localized to the nucleus. Using the testis-specific cerebroside sulfotransferase (CST) promoter and the IGF2/H19 imprinting control region (ICR), it was shown that binding of BORIS isoforms to DNA targets in vitro is methylation-sensitive and depends on the number and specific composition of ZF. The ability to bind target DNA and the presence of a specific long amino terminus (N258) in different isoforms are necessary and sufficient to activate CST transcription. Comparative sequence analyses revealed an evolutionary burst in mammals with strong conservation of BORIS isoproteins among primates. Conclusions: The extensive repertoire of spliced BORIS variants in humans that confer distinct DNA binding and transcriptional activation properties, and their differential patterns of expression among germ cells and neoplastic cells suggest that the gene is involved in a range of functionally important aspects of both normal gametogenesis and cancer development. In addition, a burst in isoform diversification may be evolutionarily tied to unique aspects of primate speciation. [ABSTRACT FROM AUTHOR]

Previously, it was shown that the CTCF paralogous gene, BORIS (brother of the regulator of imprinted sites) is expressed in male germ cells, but its function in spermatogenesis has not been defined. To develop an understanding of the functional activities of BORIS, we generated BORIS knockout (KO) mice. Mice homozygous for the null allele had a defect in spermatogenesis that resulted in small testes associated with increased cell death. The defect was evident as early as postnatal day 21 and was manifested by delayed production of haploid cells. By gene expression profiling, we found that transcript levels for Gal3st1 (also known as cerebroside sulfotransferase [CST]), known to play a crucial role in meiosis, were dramatically reduced in BORIS KO testes. We found that CST is expressed in testis as a novel testis-specific isoform, CST form FTS, that has a short exon 1f. We showed that BORIS bound to and activated the promoter of CST form FTS. Mutation of the BORIS binding site in the promoter reduced the ability of BORIS to activate the promoter. These findings define transcriptional regulation of CST expression as a critical role for BORIS in spermatogenesis. [ABSTRACT FROM AUTHOR]

Background: The canonical function of EEF1A2, normally expressed only in muscle, brain, and heart, is in translational elongation, but recent studies suggest a non-canonical function as a proto-oncogene that is overexpressed in a variety of solid tumors including breast and ovary. Transcriptional profiling of a spectrum of primary mouse B cell lineage neoplasms showed that transcripts encoding EEF1A2 were uniquely overexpressed in plasmacytomas (PCT), tumors of mature plasma cells. Cases of human multiple myeloma expressed significantly higher levels of EEF1A2 transcripts than normal bone marrow plasma cells. High-level expression was also a feature of a subset of cell lines developed from mouse PCT and from the human MM. Methodology/Principal Findings: Heightened expression of EEF1A2 was not associated with increased copy number or coding sequence mutations. shRNA-mediated knockdown of Eef1a2 transcripts and protein was associated with growth inhibition due to delayed G1-S progression, and effects on apoptosis that were seen only under serum-starved conditions. Transcriptional profiles and western blot analyses of knockdown cells revealed impaired JAK/STAT and PI3K/AKT signaling suggesting their contributions to EEF1A2-mediated effects on PCT induction or progression. Conclusions/Significance: EEF1A2 may play contribute to the induction or progression of some PCT and a small percentage of MM. Eef1a2 could also prove to be a useful new marker for a subset of MM and, ultimately, a possible target for therapy. [ABSTRACT FROM AUTHOR]

Interleukin 21 (IL-21) is a pleiotropic cytokine produced by CD4 T cells that affects the differentiation and function of T, B, and NK cells by binding to a receptor consisting of the common cytokine receptor y chain and the IL-21 receptor (IL-21R). IL-21, a product associated with IL-17-producing CD4 T cells (TH17) and follicular CD4 T helper cells (TFH), has been implicated in autoimmune disorders including the severe systemic lupus erythematosus (SLE)-like disease characteristic of BXSB-Yaa mice. To determine whether IL-21 plays a significant role in this disease, we compared IL-21 R-deficient and -competent BXSB-Yaa mice for multiple parameters of SLE. The deficient mice showed none of the abnormalities characteristic of SLE in IL-21R-competent Yaa mice, including hypergammaglobulinemia, autoantibody production, reduced frequencies of marginal zone B cells and monocytosis, renal disease. and premature morbidity. IL-21 production associated with this autoimmune disease was not a product of TH17 cells and was not limited to conventional CXCR5+ TFH but instead was produced broadly by ICOS+ CD4+ splenic T cells. IL-21 arising from an abnormal population of CD4 T cells is thus central to the development of this lethal disease, and, more generally, could play an important role in human SLE and related autoimmune disorders. [ABSTRACT FROM AUTHOR]

Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphomas showed that IRF8 transcripts were expressed at highest levels in centroblasts. either from secondary lymphoid tissue or transformed cells. In addition, staining for IRF8 was most intense in tonsillar germinal center (GC) dark-zone centroblasts. To discover B cell genes regulated by IRF8. we transfected purified primary tonsillar B cells with enhanced green fluorescent protein-tagged IRF8, generated small interfering RNA knockdowns of IRF8 expression in a mouse B cell lymphoma cell line, and examined the effects of a null mutation of IRF8 on B cells. Each approach identified activation-induced cytidine deaminase (AICDA] and BCL6 as targets of transcriptional activation. Chromatin immunoprecipitation studies demonstrated in vivo occupancy of 5′ sequences of both genes by IRF8 protein. These results suggest previously unappreciated roles for IRF8 in the transcriptional regulation of B cell GC reactions that include direct regulation of AICDA and BCL6. [ABSTRACT FROM AUTHOR]

Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes "an antibody enzyme" (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immuno-dominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP85-101 peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibody-mediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS. [ABSTRACT FROM AUTHOR]

Sixty cancer cell lines have been extensively characterized and used by the National Cancer Institute's Developmental Therapeutics Program (NCI-60) since the early 90's as screening tools for anti-cancer drug development. An extensive database has been accumulated that could be used to select individual cells lines for specific experimental designs based on their global genetic and biological profile. However, information on the human leukocyte antigen (HLA) genotype of these cell lines is scant and mostly antiquated since it was derived from serological typing. We, therefore, re-typed the NCI-60 panel of cell lines by high-resolution sequence-based typing. This information may be used to: 1) identify and verify the identity of the same cell lines at various institutions; 2) check for possible contaminant cell lines in culture; 3) adopt individual cell lines for experiments in which knowledge of HLA molecule expression is relevant. Since genome-based typing does not guarantee actual surface protein expression, further characterization of relevant cell lines should be entertained to verify surface expression in experiments requiring correct antigen presentation. [ABSTRACT FROM AUTHOR]

Transgenic mice overexpressing in B lymphocytes either Bcl-2 or a TNF receptor-associated factor (TRAF)2 mutant lacking the N- terminal R1NG and zinc finger domains located at the N terminus of the molecule (TRAF2DN), which mimics TRAF1, developed lymphadenopathy and splenomegaly due to polyclonal B cell expansion. Remarkably, TRAF2DN/Bcl-2 double-transgenic mice contained B cell populations similar to those observed in TRAF2DN mice. However, over time, they developed severe splenomegaly and lymphadenopathy, and most animals also developed leukemia, pleural effusion, and, in some cases, ascites associated with monoclonal and oligoclonal B cell neoplasms. The life span of TRAF2DN/Bcl-2 mice was markedly reduced compared with Bcl-2 and TRAF2DN single-transgenics or wild-type littermates. The expanded B cell population of TRAF2DN/Bcl-2 double-transgenic mice was primarily comprised of small/medium-size noncycling B220M/lgMH/lgDL/ CD21 L/CD23NULL/CD11b+/CD5+ cells that were Bcl-6-negative, consistent with a B-1 phenotype. The cells also expressed high levels of CD54 and other adhesion molecules. In vitro, these B cells showed comparable proliferation rates to those of wild-type counterparts but exhibited markedly increased survival and were resistant to apoptosis induced by chemotherapeutic agents and glucocorticoids. Histopathologic features were consistent with mouse small B cell lymphoma progressing to leukemia with many similarities to human chronic lymphocytic leukemia. Given that many human chronic lymphocytic leukemias overexpress TRAF1 and Bcl-2, our findings suggest that cooperation between Bcl-2 and TRAF pathways contributes to the development of this type of leukemia. [ABSTRACT FROM AUTHOR]

The WEHI 231 B cell lymphoma is used as a model of self-tolerance by clonal deletion because B cell receptor (BCR) ligation results in apoptosis. Two critical events precede cell death: an early rise and fall in expression of MYC and cell-cycle arrest associated with enhanced expression of p21, p27, and p53. CTCF is a transcription factor identified as a repressor of MYC recently shown to cause cell growth inhibition. The present studies demonstrate that BCR ligation of WEHI 231 as well as of normal immature B cells greatly increased expression of CTCF in association with down-regulation of MYC followed by growth arrest and cell death. Conditional expression of CTCF in WEHI 231 mimicked BCR ligation with activated cells showing repressed expression of MYC, enhanced expression of p27, p21, p53, and p19[SUPARF], and inhibition of cell growth and induction of apoptosis. In keeping with a central role for CTCF in control of 8 cell death, conditional expression of a CTCF antisense construct in WEHI 231 resulted in inhibition of p27, p21, p53, and p19[SUPARF] in association with enhanced expression of MYC. Activation of the endogenous CTCF locus by BCR ligation was also mimicked by three other routes to apoptotic death in WEHI 231: inhibition of the phosphoinositide 3-kinase or mTOR/FRAP signaling cascades and treatment with transforming growth factor (TGF)-β. Rapid activation of CTCF by BCR ligation or treatment with TGF-β was suppressed by ligation of CD40. These results demonstrate that CTCF is a common determinant to different pathways of death signaling in immature B cells. [ABSTRACT FROM AUTHOR]

Background: The retroviral Gag protein is the central player in the process of virion assembly at the plasma membrane, and is sufficient to induce the formation and release of virus-like particles. Recent evidence suggests that Gag may co-opt the host cell's endocytic machinery to facilitate retroviral assembly and release. Results: A search for novel partners interacting with the Gag protein of the Moloney murine leukemia virus (Mo-MuLV) via the yeast two-hybrid protein-protein interaction assay resulted in the identification of endophilin 2, a component of the machinery involved in clathrinmediated endocytosis. We demonstrate that endophilin interacts with the matrix or MA domain of the Gag protein of Mo-MuLV, but not of human immunodeficiency virus, HIV. Both exogenously expressed and endogenous endophilin are incorporated into Mo-MuLV viral particles. Titration experiments suggest that the binding sites for inclusion of endophilin into viral particles are limited and saturable. Knock-down of endophilin with small interfering RNA (siRNA) had no effect on virion production, but overexpression of endophilin and, to a lesser extent, of several fragments of the protein, result in inhibition of Mo-MuLV virion production, but not of HIV virion production. Conclusions: This study shows that endophilins interact with Mo-MuLV Gag and affect virion production. The findings imply that endophilin is another component of the large complex that is hijacked by retroviruses to promote virion production. [ABSTRACT FROM AUTHOR]

Investigates the involvement of c-MYC proto-oncogene in the expression of the gene encoding the catalytic subunit beta of protein kinase A. Induction of PKA-Cβ gene expression by c-MYC in the absence of de novo protein synthesis; Overview of soft agar colony formation by constitutive expression of PKA-Cβ; Isolation of mouse splenic B cells.

The TCL1 protooncogene is overexpressed in many mature B cell lymphomas, especially from AIDS patients. To determine whether aberrant expression promotes B cell transformation, we generated a murine model in which a TCL1 transgene was overexpressed at similar levels in both B and T cells. Strikingly, transgenic mice developed Burkitt-like lymphoma (BLL) and diffuse large B cell lymphoma (DLBCL) with attendant Bcl-6 expression and mutated JH gene segments at a very high penetrance beginning at 4 months of age. in contrast, only one mouse developed a T cell malignancy at 15 months, consistent with a longer latency for transformation of T cells by TCL1. Activation of premalignant splenic B cells by means of B cell antigen receptor (BCR) engagement resulted in significantly increased proliferation and augmented AKT-dependent signaling, including increased S6 ribosomal protein phosphorylation. Transgenic spleen cells also survived longer than wild-type spleen cells in long-term culture. Together these data demonstrate that TCL1 is a powerful oncogene that, when overexpressed in both B and T cells, predominantly yields mature B cell lymphomas. [ABSTRACT FROM AUTHOR]

Describes an interleukin-6 (IL-6) transgenic mouse model for extraosseous plasmacytoma (PCT). Incidence of PCT and cell lymphomas in IL-6-mouse; Evaluation of the transplantability of IL-6 transgenic PCT.

The mechanisms responsible for development of profound immunodeficiency and extensive lymphoproliferation that characterize infection of different species with retroviruses are only partially understood. In mice, it has been shown the activities of an unusual Gag protein are necessary and sufficient to induce these abnormalities in a syndrome designated mouse AIDS (MAIDS). Current studies suggest that complex, antigen-driven interactions between T cells and B cells result in polyclonal activation of both types of lymphocytes, aberrant cytokine production and late lymphomas. [ABSTRACT FROM AUTHOR]

Recent analysis of over 500 lymphomas occurring in NFS.V mice, congenic for Akv-type ecotropic MuLV structural genes, has revealed that about 90% are of B cell lineage as determined by demonstration of clonal rearrangements of Ig heavy chain genes, phenotyping by immunocytochemistry or cytofluorometric analysis, and by site and morphology of tumor. At least 40% of the B cell lymphomas were found to have their origin in the splenic marginal zone, a site only once before described for mouse lymphomas. Clonal somatic integrations of ecotropic MuLV occurred in 85% of tumors. [ABSTRACT FROM AUTHOR]

Comparative genome-wide expression profiling of malignant tumor counterparts across the human-mouse species barrier has a successful track record as a gene discovery tool in liver, breast, lung, prostate and other cancers, but has been largely neglected in studies on neoplasms of mature B-lymphocytes such as diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma (BL). We used global gene expression profiles of DLBCL-like tumors that arose spontaneously in Myc-transgenic C57BL/6 mice as a phylogenetically conserved filter for analyzing the human DLBCL transcriptome. The human and mouse lymphomas were found to have 60 concordantly deregulated genes in common, including 8 genes that Cox hazard regression analysis associated with overall survival in a published landmark dataset of DLBCL. Genetic network analysis of the 60 genes followed by biological validation studies indicate FOXM1 as a candidate DLBCL and BL gene, supporting a number of studies contending that FOXM1 is a therapeutic target in mature B cell tumors. Our findings demonstrate the value of the “mouse filter” for genomic studies of human B-lineage neoplasms for which a vast knowledge base already exists. [ABSTRACT FROM AUTHOR]

N-ethyl-N-nitrosourea (ENU)-induced mutagenesis provides a powerful approach for identifying genes involved in immune regulation and diseases. Here we describe a new mutant strain, HLB36S, with hereditary leukopenia. At necropsy, the mutant mice had very small thymuses and spleens. All hut the inguinal nodes were absent and there were no Peyer's patches. By flow cytometry, the ratios of T-cell subsets were normal, but B-cell development was blocked at the pre-pro-B-cell stage. The development of B1 and marginal zone B cells was relatively normal. The mutation was mapped to chromosome 3 between D3Mit221 and D3Mit224, a region that contains the Il7 gene. cDNA and genomic DNA sequences of Il7 revealed a T-to-C missense transition resulting in a change of Leu to Pro within the leader peptide that would be predicted to inhibit secretion. In keeping with this concept, we found that in vitro treatment of B-cell progenitors from mutant mice with IL-7 induced them to differentiate into pre-BII cells. Phenotypic comparisons of HLB368 with genetically targeted Il7 null mice showed many similarities along with a few differences, indicating that this ENU-induced mutant carries a novel allele. This new strain thus provides a new model for studying the functions of IL-7 on a pure C57BL/6 background. [ABSTRACT FROM AUTHOR]

Human and mouse chronic lymphocytic leukemia (CLL) develops from CD5+ B cells that in mice and macaques are known to define the distinct B1a B-cell lineage. B1a cells are characterized by lack of germinal center (GC) development, and the B1a cell population is increased in mice with reduced GC formation. As a major mediator of follicular B-cell migration, the G protein-coupled receptor Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) directs B-cell migration in the lymphoid follicles in response to its endogenous ligands, oxysterols. Thus, upregulation of EBI2 drives the B cells toward the extrafollicular area, whereas downregulation is essential for GC formation. We therefore speculated whether increased expression of EBI2 would lead to an expanded B1 cell subset and, ultimately, progression to CLL. Here, we demonstrate that B-cell-targeted expression of human EBI2 (hEBI2) in mice reduces GC-dependent immune responses, reduces total immunoglobulin M (IgM) and IgG levels, and leads to increased proliferation and upregulation of cellular oncogenes. Furthermore, hEBI2 overexpression leads to an abnormally expanded CD5+ B1a B-cell subset (present as early as 4 days after birth), late-onset lymphoid cancer development, and premature death. These findings are highly similar to those observed in CLL patients and identify EBI2 as a promoter of B-cell malignancies. [ABSTRACT FROM AUTHOR]

The serine-threonine kinase ataxia-telangiectasia mutated (ATM) plays a central role in maintaining genomic integrity. In mice, ATM deficiency is exclusively associated with T-cell lymphoma development, whereas B-cell tumors predominate in human ataxia-telangiectasia patients. We demonstrate in this study that when T cells are removed as targets for lymphomagenesis and as mediators of immune surveillance, ATM-deficient mice exclusively develop early-onset immunoglobulin M+ B-cell lymphomas that do not transplant to immunocompetent mice and that histologically and genetically resemble the activated B cell-like (ABC) subset of human diffuse large B-cell lymphoma (DLBCL). These B-cell lymphomas show considerable chromosomal instability and a recurrent genomic amplification of a 4.48-Mb region on chromosome 18 that contains Malt1 and is orthologous to a region similarly amplified in human ABC DLBCL. Of importance, amplification of Malt1 in these lymphomas correlates with their dependence on nuclear factor (NF)-κB, MALT1, and B-cell receptor (BCR) signaling for survival, paralleling human ABC DLBCL. Further, like some human ABC DLBCLs, these mouse B-cell lymphomas also exhibit constitutive BCR-dependent NF-κB activation. This study reveals that ATM protects against development of B-cell lymphomas that model human ABC DLBCL and identifies a potential role for T cells in preventing the emergence of these tumors. [ABSTRACT FROM AUTHOR]

Basophils and mast cells play critical roles in host defense against pathogens and allergic disorders. However, the molecular mechanism by which these cells are generated is not completely understood. Here we demonstrate that interferon regulatory factor-8 (IRF8), a transcription factor essential for the development of several myeloid lineages, also regulates basophil and mast cell development. Irf8-/- mice displayed a severe reduction in basophil counts, which was accounted for by the absence of pre-basophil and mast cell progenitors (pre-BMPs). Although Irf8-/- mice retained peripheral tissue mast cells, remaining progenitors from Irf8-/- mice including granulocyte progenitors (GPs) were unable to efficiently generate either basophils or mast cells, indicating that IRF8 also contributes to the development of mast cells. IRF8 appeared to function at the GP stage, because IRF8 was expressed in GPs, but not in basophils, mast cells, and basophil/mast cell-restricted progenitor cells. Furthermore, we demonstrate that GATA2, a transcription factor known to promote basophil and mast cell differentiation, acts downstream of IRF8. These results shed light on the pathways and mechanism underlying the development of basophils and mast cells. [ABSTRACT FROM AUTHOR]

B cell lymphoma 6 (BCL6) corepressor (BCOR) was discovered as aBCL6-interacting corepressor, but little is known about its other biological activities in normal B cell development and unction. Previously, we found that interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein, directly targets a large number of genes in germinal center B cells including BCL6. In this study, we screened potenial binding partners of IRF8 using a retrovirus-based protein complementation assay screen in a mouse pre-B cell line. We found that IRF8 interacts directly with BCOR and that the α-helical region of IRF8 and the BCL6 binding domain of BCOR are required for this interaction. In addition, IRF8 protein interacts directly with BCL6. Using an siRNA-mediated IRF8 knockdown mouse B cell lymphoma cell line, we showed that IRF8 represses Bcor and enhancesBcl6 transcription. Taken together, these data suggest that a complex comprising BCOR-BCL6-RF8 modulates BCL6-associated transcriptional regulation of germinal center B cell function. [ABSTRACT FROM AUTHOR]

The main function of the 3'-5' DNA exonuclease TREX1 is to digest cytosolic single-stranded DNA to prevent activation of cell-intrinsic responses to immunostimulatory DNA. TREX1 translocates to the nucleus following DNA damage with its nuclear activities being less well defined. Although mutations in human TREX1 have been linked to autoimmune/inflammatory diseases, the mechanisms contributing to the pathogenesis of these diseases remain incompletely understood. Here, using mass spectrometry and co-immunoprecipitation assays and in vivo overexpression models, we show that TREX1 interacts with poly(ADP-ribose) polymerase-1 (PARP1), a nuclear enzyme involved in the DNA damage response. Two zinc finger domains at the amino terminus of PARP1 were required for the interaction with TREX1 that occurs after nuclear translocation of TREX1 in response to DNA damage. Functional studies suggested that TREX1 may contribute to stabilization of PARP1 levels in the DNA damage response and its activity. These results provide new insights into the mechanisms of singlestranded DNA repair following DNA damage and alterations induced by gene mutations. [ABSTRACT FROM AUTHOR]